This invention relates to thermostable DNA polymerases useful for DNA sequencing.
Innis et al., Proc. Natl. Acad. Sci. USA 85:9436-9440, 1988 state that a DNA polymerase from Thermus aquaticus (termed Taq or Taq DNA polymerase) is useful for DNA sequencing.
Lawyer et al., J. Biol. Chem. 264:6427, 1989 describe the isolation and cloning of DNA encoding Taq. The DNA and amino acid sequences described in this publication define the Taq gene and Taq DNA polymerase as those terms are used in this application.
Gelfand et al., U.S. Pat. No. 4,889,818, describe the isolation and expression of Taq and state that:
It has also been found that the entire coding sequence of the Taq polymerase gene is not required to recover a biologically active gene product with the desired enzymatic activity. Amino-terminal deletions wherein approximately one-third of the coding sequence is absent have resulted in producing a gene product that is quite active in polymerase assays. PA1 Thus, modifications to the primary structure itself by deletion, addition, or alteration of the amino acids incorporated into the sequence during translation can be made without destroying the activity of the protein. PA1 In the particular case of Taq polymerase, evidence indicates that considerable deletion at the N-terminus of the protein may occur under both recombinant and native conditions, and that the activity of the protein is still retained. It appears that the native proteins isolated may be the result of proteolytic degradation, and not translation of a truncated gene. The mutein produced from the truncated gene of plasmid pFC85 [containing a 2.8 kb HindIII-Asp718 restriction fragment; where the HindIII site is at codons 206 and 207] is, however, fully active in assays for DNA polymerase, as is that produced from DNA encoding the full-length sequence. Since it is clear that certain N-terminal shortened forms are active, the gene constructs used for expression of the polymerase may also include the corresponding shortened forms of the coding sequence.